Designing Novel Protein Assemblies as Rigid Symmetric Scaffolds for Cryo-EM Imaging

总结 低温电子显微镜（cyro-EM）正在经历非凡的技术革命，使其成为 可以对大的蛋白质组装体和核酸复合物进行原子细节的成像。然而，关键的是， 该方法主要适用于大型组件（尤其是对称的组件），而较小的 蛋白质（例如小于50 kDa）低于当前的技术限制。考虑到平均细胞 蛋白质比这小，迫切需要新的分子策略，以充分实现 cryo-EM的转化潜力。 人们早就认识到，冷冻电镜可以观察到更典型大小的较小蛋白质 如果它们可以在更大的分子支架上刚性排列，以提供质量和更高的对比度， 理想地以赋予高度对称性的方式。这一目标在很大程度上一直难以实现。两个关键 挑战在于（1）如何以刚性方式将蛋白质附着到支架上， （2）如何开发一个模块化系统，不需要 不可预测的分子工程和优化以及对每个新目标的费力的EM测试 分子进行研究。 该技术开发提案回答了开发冷冻EM支架的挑战， 是对称的，模块化的，刚性的，通过应用我们小组和合作者的新方法来设计 精确定义和高度对称的蛋白质组装体。设计的蛋白质笼提供了对称的 用于外部附件的核心。我们将称为DARPins的蛋白质与这些对称核心进行基因融合， 其已经被开发为用于特异性和刚性结合不同蛋白质的简易系统， 在实验室里进化它们的环序列。重要的是，DARPin和 通过使用连续α螺旋融合方法， 设计新型蛋白质笼的想法。 在初步工作中，我们评估了第一代模块化脚手架候选人之一， EM.当单独成像时（笼形磁芯加DARPin），磁芯分辨率为3.1 nm，DARPin保持不变 刚性足以在中等分辨率（约3.5至5.5 μ m）下看到。在新的初步数据中， 候选物已经用结合的第一货物蛋白GFP成像。DARPin在那里有些僵化 与游离形式相比，虽然结合的GFP本身整体上以4.7 μ m的分辨率可视化，但新的 小蛋白质冷冻电镜的基准。这些实验展示了最有前途和最模块化的 到目前为止，小蛋白质成像的路线，同时也显示了需要进一步的设计步骤才能达到 近原子分辨率本研究将设计、评价和完善一套新型的冷冻电镜支架 基于设计的蛋白质笼，对结构生物学和医学产生了重大影响。

SUMMARY Cryo-electron microscopy (cyro-EM) is undergoing extraordinary revolutions in technology, making it possible to image large protein assemblies and nucleic acid complexes in atomic detail. Critically however, the methods are mainly applicable to large assemblies (especially those that are symmetric), while smaller proteins (e.g. smaller than 50 kDa) are below current technological limits. Given that the average cellular protein is smaller than that, new molecular strategies are needed badly in order to fully realize the transformational potential of cryo-EM. It has long been recognized that smaller proteins of more typical size could be visualized by cryo-EM if they could be arrayed rigidly on a larger molecular scaffold to provide mass and higher contrast, and ideally in a way that would confer a high degree of symmetry. This goal has been largely elusive. Two key challenges have been (1) how to attach proteins to scaffolds in a rigid way so that important imaging advantages could be exploited, and (2) how to develop a modular system that would not require unpredictable molecular engineering and optimization and laborious EM testing for every new target molecule to be studied. This Technology Development proposal answers the challenge of developing cryo-EM scaffolds that are symmetric, modular, and rigid, by applying new methods from our group and collaborators for designing precisely defined and highly symmetric protein assemblies. Designed protein cages provide a symmetric core for external attachments. To these symmetric cores, we genetically fuse proteins known as DARPins, which have been developed as facile systems for binding specifically and rigidly to diverse proteins based on laboratory evolution of their loop sequences. Importantly, the connection between the DARPin and the designed symmetric core is made relatively rigid by using a continuous alpha helical fusion approach, an idea introduced earlier for designing novel protein cages. In preliminary work we evaluated one of the first-generation modular scaffold candidates by cryo- EM. When imaged by itself (cage core plus DARPin) the core is resolved to 3.1Å and the DARPin is held rigidly enough to see at medium resolution (about 3.5 to 5.5 Å). In new preliminary data the first scaffold candidate has been imaged with a first cargo protein, GFP, bound. There the DARPin is somewhat rigidified compared to the free form, while the bound GFP itself is visualized at 4.7 Å resolution overall, a new benchmark for cryo-EM of small proteins. These experiments lay out the most promising and most modular route so far for imaging small proteins, while also showing what further design steps are required to reach near-atomic resolution. This leading work will design, evaluate and perfect a novel set of cryo-EM scaffolds based on designed protein cages, with major impacts on structural biology and medicine.